Protein analysis was performed with Western blot after 72h of miRNA or siRNA transfection, unless indicated differently (Details in Supplemental Materials; Primary antibodies in Supplementary Table 7 ). Secondary IRDye®680 or IRDye®800-conjugated antibodies (LI-COR, Lincoln, NE, USA) were used for band visualization. Membranes were scanned and analyzed with Odyssey scanner and Odyssey 2.1, respectively (LI-COR, Lincoln, NE, USA). For quantification, local background subtraction and β-Actin normalization was performed.
Odyssey 2
Manufactured by LI COR
Sourced in United States
The Odyssey 2.1 software is a data analysis tool developed by LI-COR. It provides a platform for managing and interpreting data generated from LI-COR's imaging systems and plate readers. The software enables users to perform various analytical functions, such as quantification and visualization of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (8 mentions)
Odyssey scanner, by LI COR (4 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Irdye 800, by LI COR (1 mentions)
Irdye 680, by LI COR (1 mentions)
Odyssey reader, by LI COR (1 mentions)
Beckman l8 70m ultracentrifuge, by Beckman Coulter (1 mentions)
Anti actin clone c4, by MP Biomedicals (1 mentions)
Sc 718, by Santa Cruz Biotechnology (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Odyssey ir imaging system, by LI COR (1 mentions)
Gapdh, by Abcam (1 mentions)
4 20 tris hcl ready gels, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
T per reagent, by Thermo Fisher Scientific (1 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ab104225, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
25 protocols using odyssey 2
1
Protein Analysis via Western Blot after miRNA/siRNA Transfection
2
Cytokine Profiling of Mesenchymal Stem Cell Secretome
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MSCs were transfected for 48h, starved overnight (o.n.), and grown in starvation medium for 72h if not indicated differently. Breast cancer or epithelial cells were grown for 72h in complete growth medium. Subsequently, CM was retrieved, cell debris was removed by centrifugation and stored immediately at -80°C and thawed only once for experimental usage. For ultracentrifugation (UC), the CM was first centrifuged for 30min at 2,000g to remove dead cells and cell debris. The residual supernatant was centrifuged at 100,000g for 180min using a SW 41 Ti Rotor in a Beckman L8-70M Ultracentrifuge (Beckman Coulter, California, USA). Separation of proteins larger than 4kDa in CM of cancer cells was performed with AMICON® Ultra-4 filtration units (Merck Millipore, Darmstadt, Germany) at 4000g for 30 minutes.
MSC CM was analyzed for secreted proteins using Human Cytokine Array Kit, Panel A (R&D Systems, Wiesbaden-Nordenstadt, Germany) according to manufacturer's instructions except that IRDye®800CM Streptavidin (LI-COR, Lincoln, NE, USA) was used in a 1:4000 dilution in PBS for visualization. Membranes were scanned with Odyssey Reader and analyzed with Odyssey 2.1 (LI-COR, Lincoln, NE, USA). Fluorescence linked-immunosorbent assay (FLISA) was used for protein quantification in CM (Protocol in Supplemental Materials).
MSC CM was analyzed for secreted proteins using Human Cytokine Array Kit, Panel A (R&D Systems, Wiesbaden-Nordenstadt, Germany) according to manufacturer's instructions except that IRDye®800CM Streptavidin (LI-COR, Lincoln, NE, USA) was used in a 1:4000 dilution in PBS for visualization. Membranes were scanned with Odyssey Reader and analyzed with Odyssey 2.1 (LI-COR, Lincoln, NE, USA). Fluorescence linked-immunosorbent assay (FLISA) was used for protein quantification in CM (Protocol in Supplemental Materials).
3
Western Blot Quantification of Protein Abundance
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Protein abundance was quantified with western blotting. Cells were seeded into 6-well plates and proteins were isolated the following day at 80% confluency. Primary antibodies used were Anti-Actin (clone C4) (MP Biomedicals GmbH, Eschwege, Germany) for β-Actin (1:10,000 dilution) and SC-718 (Santa Cruz Biotechnology, Inc., Heidelberg, Germany) for cyclin D1 (1:200 dilution). Bands were visualized with secondary IRDye®680 and IRDye®800-conjugated antibodies and membranes were scanned with Odyssey scanner and analyzed with Odyssey 2.1 (LI-COR, Lincoln, NE, USA).
4
Quantitative Western Blot Analysis
5
Western Blot Analysis of Cell Extracts
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Following treatments, cells were washed in ice-cold PBS-CM (PBS with 1 mM MgCl2 and 0.1 mM CaCl2) and whole cell extracts were prepared in Tris Lysis Buffer (50 mm Tris-Cl, pH 7.4, 150 mm NaCl, 1% NP-40, 0.5% sodium deoxycholate, 5 mm EDTA, 5 mm EGTA, 1X Complete™ protease inhibitor cocktail, 1 mM phenylmethylsulfonyl fluoride). Proteins in cell extracts or immunoprecipitates were prepared in 1X Laemmli sample buffer (Bio-Rad) supplemented with 50 mM dithiothreitol, heated for 5 min at 96°C and loaded for electrophoresis onto 8% SDS-polyacrylamide gels or 4%–12% Tris/HEPES-SDS Bolt™ precast gels (Thermo-Invitrogen). Size-separated proteins were transferred to nitrocellulose membranes (Bio-Rad). Primary antibodies (described above) and IRdye800-conjugated secondary antibodies were used to detect target proteins using the LI-COR Odyssey infrared imaging system (LI-COR Biosciences). Quantification of the intensity of the bands was obtained using Odyssey 2.0 software (LI-COR Biosciences).
6
Chemotaxis and Immunoblot Assay Analysis
7
Western Blot Protein Detection
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Samples were loaded onto 8% SDS-polyacrylamide gels or 4–12% Tris/HEPES-SDS BoltTM precast gels (Thermo-Invitrogen) for electrophoresis. The size-separated proteins were then transferred to nitrocellulose membranes (Bio-Rad). Primary antibodies (described above) and IRdye800-conjugated secondary antibodies were used to detect target proteins. Signal was detected using the LI-COR Odyssey IR imaging system (LI-COR Biosciences). Quantification of the intensity of the bands was obtained using Odyssey 2.0 software (LI-COR Biosciences).
8
Western Blot Analysis of NeuN Proteins
9
Nuclear Translocation of NF-κB by Mn and MitoQ
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To examine nuclear translocation of p65 NF-κB by Mn and Mn + MitoQ, cells were exposed to Mn doses (0, 10 and 100 μM for 5 h) with or without prior treatment with 1 μM MitoQ or vehicle control for 30 min. Post-exposure, cells were isolated for subcellular fractionation using a nuclear and cytoplasmic extraction kit (Thermo Scientific, Rockford, IL, USA). Isolated fractions were confirmed by Western blotting using antibodies against p65 NF-κB. Controls were determined by probing nuclei for lamin A/C. All antibodies were obtained from Cell Signaling Technology, Boston, MA, USA. An IRDye 800 conjugated affinity purified anti-rabbit (Rockland Immunochemicals, Gilbersville, PA, USA) or Alexa-Fluor-680-conjugated anti-mouse (Invitrogen, Waltham, MA, USA) was used as secondary antibody. Bands were visualized using an Odyssey scanner (Li-Cor Biosciences, Lincoln, NE, USA) and Odyssey 2.1 software (Li-Cor, Lincoln, NE, USA) and quantified using ImageJ v1.48.
All statistical analyses, boxplots and heatmaps were generated in R v3.2.3. Schematic figures were created using online software atBioRender.com . Abbreviations used throughout the text are provided in Supplementary Text S1 .
All statistical analyses, boxplots and heatmaps were generated in R v3.2.3. Schematic figures were created using online software at
10
Quantifying Hog1 Phosphorylation in Yeast
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Yeast cells were grown to mid-log phase in YPD liquid medium. Flu was added to the medium, and 1 ml aliquots were withdrawn at the indicated time points. Cells were resuspended in sodium dodecyl sulphate (SDS) buffer (50 mM Tris-HCl [pH 6.8], 10% Glycerol, 2% SDS, 5 mM NaF, 1 mM Na3VO4 and 5% β-mercaptoethanol), boiled for 10 min and centrifuged at 13 000 x g for 10 min to obtain protein extracts. Protein concentration was measured using the RC DC Protein Assay kit (Bio-Rad), and 20 μg of total protein was loaded onto 10% Mini-PROTEAN TGX precast gels (Bio-Rad) and blotted on nitrocellulose membranes (Bio-Rad). Phosphorylated Hog1 was detected using anti-phospho-p38 MAPK antibody (Cell Signaling Technology) with 1:2000 dilution and IRDye 800CW donkey antibody against rabbit IgG (LI-COR Biosciences) with 1:5000 dilution. Total Hog1 protein was detected using anti-Hog1 yC-20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) with 1:2000 dilution and IRDye 680RD donkey antibody against goat IgG (LI-COR Biosciences) with 1:5000 dilution. Signals were detected using the Odyssey Infrared Imaging System and quantified using the Odyssey 2.1 software (LI-COR Biosciences). All phosphorylated Hog1 values were normalized against the 5-min sample taken from the control strain.
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